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. Author manuscript; available in PMC: 2022 Jul 18.
Published in final edited form as: Nat Immunol. 2020 Jun 22;21(7):756–765. doi: 10.1038/s41590-020-0698-1

Fig. 5: HDM-induced IL-33 is dependent on SAA1 dissociation.

Fig. 5:

(a) Sequence of SAA1. Indicated are: secondary structure α-helices (α 1–4) and loops (connecting lines between α-helices), C-terminal tail (CTL). Amino acids located within the hydrophobic core of the SAA1 hexamer are shaded in green. The epitopes recognized by affinity-purified IgGs raised against residues 27–44, 40–63, 68–84, and 89–104 are indicated by yellow lines (used in panel e). The mutation site at the hydrophobic core is indicated by an orange dot (used in panel f). C-terminal deletion of amino acids (Δ1–11) is shaded in blue. (b) BEAS-2B supernatants were cleared of lipid-bound SAA1 pulling down HDL-bound SAA1 using a polyclonal goat anti-ApoA1 antibody. Cleared supernatants were immunoblotted with a monoclonal mouse anti-human SAA1 antibody (Acris). (c) IL-33 secretion induced by rSAA1 alone (open bars) or in complex with a mouse monoclonal antibody specific for human SAA1 (αSAA ab, closed bars) in the BEAS-2B cell line. (d) Supernatants of cells left either untreated (media) or stimulated with recombinant SAA1 (rSAA) and immunoblotted with a monoclonal antibody specific for human SAA (R&D Systems; MAB30196). (e) IL-33 release from BEAS-2B cells after incubation with sequence-specific rabbit antisera specific for human SAA1 (**P = 0.0029). (f) HDM-triggered IL-33 release in BEAS-2B cells transfected with empty plasmid control (EV), wildtype SAA1 (WT) overexpression plasmid or a SAA1 plasmid with a Trp to Ala mutation at position 53 of the amino acid sequence (W53A) to mutate the hydrophobic core of SAA1 (WT to EV **P = 0.0032; WT to W53A **P = 0.0085). (g) HDM-induced IL-33 amounts in epithelial cells grown in media supplemented with charcoal-stripped FBS (**P = 0.0027). Cropped images are shown. Data shown as means ± SEM represent are representative of 2 independent experiments (c, d, f, g) or 3 (e) each containing at least n=4 biologically independent samples. Blots are representative of 2 (d) and 4 (b) independent experiments. P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (e, g) or Dunett’s post hoc analysis (f). ****P ≤ 0.0001